This proposal is focussed on the study of two bacterial respiratory oxidases. A combination of biochemical, biophysical and genetics techniques will be employed to explore how these two membrane-bound enzymes function to reducer molecular oxygen to water and, concomitantly, to translocate protons across the bacterial membrane to generate a proton motive force. The bd-type ubiquinol oxidase of E. coli contain two subunits and three heme prosthetic groups. Several oxygenated forms of this enzyme have been spectroscopically characterized under equilibrium conditions. One of our goals is to monitor the time evolution of these forms during a single turnover of the oxidase using flow-flash transient kinetics techniques. Studies such as this with the native oxidase are absolutely essential to provide the background information and the spectroscopic techniques which are essential in order to properly utilize mutagenesis as a tool for addressing questions of structure and function. Genetics methods along with biochemical techniques have defined regions of the polypeptides that are involved in heme binding, ubiquinol binding, and possibly, subunit interaction. These studies will be extended to define additional residues that may be critically involved in the active sites. A variety of techniques will be utilized in the characterization of mutants, including resonance Raman, electron spin resonance, and Fourier transform infrared spectroscopies. Our working model places the two active sites of this enzyme, for quinol oxidation and for the reduction of oxygen to water, on opposite sides of the membrane. The net proton translocation across the membrane which is observed during enzyme turnover can be explained by the chemistry occurring at these two separated active sites, without the need to postulate a proton-conducting channel. This model will be further tested. A second project is to examine the aa(3)-type cytochrome c oxidase of Rb. sphaeroides. This enzyme is closely related to the eukaryotic cytochrome c oxidase. We have developed the techniques required to evaluate structural perturbations on each of the metal redox centers within subunit I (heme a, heme a(3) and Cu(B)) caused by specific amino acid substitutions. The consequences of specific mutations on both proton pumping and electron transfer can also be quantified. Mutations will be made in selected residues that are highly conserved in this family of oxidases in order to identify residues critical for heme and Cu binding and also for proton movements during turnover. The aa(3)-type oxidase must have a proton-conducting channel spanning the membrane. Among our targets will residues which are components of the channel.